Relationship between intrathrombotic appearance of HSP27 and HSP70 and thrombus ages in a murine model of deep vein thrombosis

Heat shock proteins (HSPs) are molecular chaperones whose primary function is cytoprotection, supporting cell survival under (sub) lethal conditions. They have been implicated in various diseases such as inflammatory diseases and cancer due to their cytoprotective and immunomodulatory effects, and their biological mechanisms have been studied. Central family members include, HSP27, which is induced by various stimuli such as heat shock, hypoxia, hyperoxia, ultraviolet exposure, and nutritional deficiency, and HSP70, which is homeostatically expressed in many organs such as the gastrointestinal tract and has anti-cell death and anti-inflammatory effects. In this study, HSP27 and HSP70 were investigated during thrombus formation and dissolution in a deep vein thrombosis model by immunohistochemistry to determine their involvement in this process and whether their expression could be used as a forensic marker. In the process of thrombus formation and lysis, HSP27 and HSP70 were found to be expressed by immunohistochemical analysis. The role of inhibitors of HSP27 and HSP70 in the pathogenesis of thrombosis in mice was also investigated. When HSP27 or HSP70 inhibitors were administered, thrombi were significantly smaller than in the control group on day 5 after inferior vena cava ligation, indicating pro-thrombotic effects HSP27 and HSP70. If HSP27- or HSP70-positive cells were clearly visible and easily identifiable in the thrombus sections, the thrombus was presumed to be more than 10 days old. Thus, the detection of intrathrombotic HSP27 and HSP70 could forensically provide useful information for the estimation of thrombus ages. Collectively, our study implied that both HSP27 and HSP70 might be molecular targets for thrombus therapy and that the detection of HSP-related molecules such as HSP27 and HSP70 could be useful for the determination of thrombus ages.


Intrathrombotic appearance of HSP27 + or HSP70 + cells
In the sub-acute and chronic phase thrombi, the HSP27 + and HSP70 + cells were detected (Fig. 1c and Table 1).Until day 10, when the inflammatory response is dominant in thrombus formation 25 , the number of HSP27 + cells exceeded the number of HSP70 + cells (Fig. 4).The HSP27 + cells were more abundant than HSP70 + cells until the thrombus entered the chronic phase, and by day 10, HSP27 + cells were significantly more numerous than HSP70 + cells, but by day 14, both HSP27 and HSP70 were almost the same.These results suggest that the kinetics of HSP27 and HSP70 expression in thrombus can be a useful index for determining the degree of obsolescence of thrombus even in autopsy cases.

Both HSP27 and HSP70 inhibitors reduced thrombi size in mice
In another experiment, mice were treated with HSP27 or HSP70 inhibitors to explore the role of HSPs.Five days after IVC ligation, mice treated with pharmacological inhibitors of HSP27 or HSP70 developed smaller thrombi compared to PBS-treated control mice (Fig. 5a,b,d,e).Furthermore, blood flow was increased in mice treated with inhibitors compared to control mice (Fig. 5c and f).These observations suggest that HSP27 and HSP70 may play an important role in the pathogenesis of thrombosis.

Estimation of thrombus age using human samples
In the next step, thrombus samples collected from human were analyzed histopathologically and immunohistochemically to estimate thrombus age (No. 1 to 7 in Fig. 6 and Table 2).As an example, No.1, the thrombus showed that the collagen content area was approximately 45% and number of hemosiderinpositive cells was 33 (Fig. 6a and Table 2).In addition, the ratio of MPO-positive neutrophils to CD68-positive macrophages (N/M ratio) referred to 0.9 (Fig. 6a and Table 2).Moreover, the number of HSP70-positive cells was markedly lower than that of HSP27-positive cells.(Fig. 6a and Table 2) Taken together, these observations suggest that the human thrombus No. 1 may be 7-10 days old based on our findings 25,26 .The thrombi from No. 2 to 7 were analyzed in the same way and the thrombus age was estimated (Fig. 6b-g and Table 2).

Discussion
The expression of HSPs in the lungs, myocardium, and kidneys under heat stress was clear, and their expression was a useful indicator for estimating the cause of death and time since death [22][23][24] .To elucidate the types of stresses under which HSPs are expressed, we confirmed their expression in experimental thrombi.HSPs were identified not only in experimental thrombi but also in thrombi found postmortem, suggesting that the relationship between thrombi and HSPs is also an important factor.In fact, the degree of thrombus patency was closely related to HSP expression.
In this study, we performed immunohistochemical search for heat shock proteins, especially HSP27 and HSP70.HSP27 is ubiquitously present in both the cytoplasm and nucleus, and its expression level increases upon exposure to heat shock and various stress conditions.The major roles of HSP27 include regulation of protein folding by chaperone function, protein degradation, maintenance of the cytoskeleton, regulation of the cell cycle, immune response, promotion of cancer, induction of resistance to anticancer drugs, aging, biomarkers of disease, exacerbation of neurodegenerative diseases, development, and differentiation [6][7][8][9][27][28][29][30][31][32][33][34][35][36] . HSP70sare abundant in cancer cells and have been shown to suppress multiple apoptotic pathways, regulate necrosis, evade cellular senescence programs, interfere with tumor immunity, promote angiogenesis, and even affect tumor metastasis 6,8,30 .
Recently, Thienel et al. reported that distinct antithrombotic patterns were detected in the platelets of hibernating brown bears.Among these, HSP47, which is an essential intracellular chaperone molecule for facilitating collagen production within cells that secrete collagen, showed the most notable decrease.Lowering or eliminating HSP47 led to the inhibition of immune cell activation and the formation of neutrophil extracellular traps, contributing to the prevention of blood clotting in bears, patients with spinal cord injuries, and mice 37 .In addition, HSP47 was reported to be present on the platelet surface, where it interacts with collagen, stabilizes platelet adhesion, and increases collagen-mediated signaling, thus promoting thrombus formation and hemostasis 38 .On the other hand, ex vivo, pharmacological inhibition of Hsp70 in human whole blood prevented the formation of platelet aggregates on collagen under shear 39 .In fact, we found that both HSP70 and HSP27 inhibitors showed reduced thrombi mass in mice.These findings of these HSPs could serve as the foundation for a new protective mechanism against the development of thrombus formation.
We found a relationship between the degree of thrombosis and the expression of HSPs in a DVT model.Although few positive cells and gene expressions were observed until the third day of thrombus formation, both HSP27 and HSP70 increased from the 5-day to the 10-day, suggesting that the stress of IVC ligation and the subsequent initiation of blood coagulation induced these HSPs expressions.The intrathrombotic HSPs were mainly expressed on F4/80 + macrophages to promote thrombolysis, and were involved in the formation of intrathrombotic neovessels.Although the expression period of HSPs was short, ranging from 5 to 14 days, HSP27 was expressed in large numbers and clearly, especially on 10-day, and may be a useful indicator to determine the age of thrombosis.
Taken together, our observations implied that both HSP27 and HSP70 might be a molecular target for thrombus therapy from the aspects of clinical relevance.Moreover, the detection of intrathrombotic HSP27 and HSP70 could provide useful information for the estimation of thrombus ages with potential applications in forensics.Collectively, our findings support the interdisciplinary, multifaceted nature of both HSP27 and HSP70.

Stasis-induced deep vein thrombus model
Specific pathogen-free 8-to 10-week-old male BALB/c mice were purchased from SLC (Shizuoka, Japan).Intravenous thrombus was induced as described previously [11][12][13][14] .Under the deep anesthesia by isoflurane inhalation (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), an incision was made on the abdominal wall, and the inferior vena cava (IVC) was exposed and ligated.At the indicated time intervals, mice were euthanized by the inhalation of over-dose isoflurane, and thrombus samples were obtained.We used 5 mice at each time point.In another series, BALB/c mice were treated intraperitoneally HSP27 inhibitor (50 μg/mouse/ day; HY-124653, MedChemExpress, Monmouth Junction, NJ) or HSP70 inhibitor (0.1 mM/mouse/day; A18734, Adooq BioScience, Irvine, CA), or PBS as controls at one day before the IVC ligation and at 1 and 3 days after the IVC ligation.At day 5 after the IVC ligation, intravenous thrombi were harvested for the determination of the weights and blood flow.Five to six mice were used per group.All animal experiments were approved by the Committee on Animal Care and Use of Wakayama Medical University.

Human deep vein thrombus samples
A total of 7 human thrombus samples were collected from forensic autopsy cases with a postmortem interval (PMI) of less than 72 h.The individual ages ranged from 33 to 90 years (mean age, 65.1 years).Samples were subjected to subsequent analyses.

Figure 4 .
Figure 4. Changes in intrathrombotic HSP27 + cell and HSP70 + cell numbers after IVC ligation.Both positive cell number of HSP27 and HSP70 showed maximum values on day 10, but HSP27 was significantly abundant than HSP70.n = 5 in each group.

Figure 5 .
Figure 5.The effects of HSP27 and HSP70 inhibitor in murine thrombosis.(a) Macroscopic appearance of venous thrombi obtained from mice treated with HSP27 inhibitor or PBS as controls.Representative results from five independent animals are shown.Thrombus mass (b) and thrombosed blood flow (c) were measured at 5 days after IVC ligation.(d) Macroscopic appearance of venous thrombi obtained from mice treated with HSP70 inhibitor or PBS as controls.Representative results from six independent animals are shown.Thrombus mass (e) and thrombosed blood flow (f) were measured at 5 days after IVC ligation.All values represent the mean ± SEM (n = 5-6 animals).**p < 0.01, *p < 0.05 vs. control.